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Methods in Molecular Biology (Clifton,... 2014In vivo lineage tracing is a valuable technique to study cellular behavior. Our lab developed a lineage tracing method, based on the Cre/lox system, to genetically...
In vivo lineage tracing is a valuable technique to study cellular behavior. Our lab developed a lineage tracing method, based on the Cre/lox system, to genetically induce clonal labelling of cells and follow their progeny. Here we describe a protocol for temporally controlled clonal labelling and for microdissection of individual mouse hair follicles. We further present staining and visualization techniques used in our lab to analyze clones issued from genetically induced labelling.
Topics: Animals; Cell Lineage; Clone Cells; Corn Oil; Genes, Reporter; Glycerol; Hair Follicle; Injections; Integrases; Luminescent Proteins; Mice; Microdissection; Microscopy, Confocal; Molecular Imaging; Staining and Labeling; Tamoxifen; Tissue Fixation
PubMed: 24281870
DOI: 10.1007/7651_2013_48 -
Stem Cells (Dayton, Ohio) Jun 2007Human embryonic stem cells (hESCs) possess unique properties for studying mechanisms controlling cell fate commitment during early mammalian development. Gain of...
Human embryonic stem cells (hESCs) possess unique properties for studying mechanisms controlling cell fate commitment during early mammalian development. Gain of function is a common strategy to study the function of specific genes involved in these mechanisms. However, transgene toxicity can be a major limitation, especially with factors influencing proliferation or differentiation. Here, we describe an efficient method based on the inducible recombinase Cre-ERT2 for conditional gene expression in hESCs and their differentiated derivatives. Using this approach, we have established several hESC sublines inducible for the expression of the enhanced green fluorescent protein and the transforming growth factor beta family member Nodal. Together, these results demonstrate that Cre-ERT2 can be used to control gene expression in undifferentiated and differentiated cells, thereby providing the first conditional transgene expression system that works effectively in hESCs. Disclosure of potential conflicts of interest is found at the end of this article.
Topics: Cells, Cultured; Embryonic Stem Cells; Gene Expression Regulation; Gene Transfer Techniques; Green Fluorescent Proteins; Humans; Integrases; Nodal Protein; Organisms, Genetically Modified; Protein Structure, Tertiary; Receptors, Estrogen; Tamoxifen; Transforming Growth Factor beta; Transgenes
PubMed: 17322103
DOI: 10.1634/stemcells.2006-0825 -
Molecular Therapy : the Journal of the... Jan 2004Systemic administration of therapeutic proteins has value in treating a wide variety of disorders, including erythropoietin (Epo)-responsive anemias. Recombinant...
Systemic administration of therapeutic proteins has value in treating a wide variety of disorders, including erythropoietin (Epo)-responsive anemias. Recombinant proteins, however, are costly and require repeated injections, while gene delivery approaches have suffered from inefficiency and difficulties with regulation. The skin effectively delivers polypeptides to the circulation, and improved approaches would support sustainable, topically regulated protein expression after a single vector injection. Toward this goal, we generated lentivectors in which both gene delivery and persistence in skin are regulated by administration of distinct steroid ligands. Following a single injection of regulated lentivector into human skin regenerated on immunodeficient mice, topical glucocorticoid ligands regulated Epo levels and hematocrit over time. Abrogation of gene delivery was achieved by both glucocorticoid cessation and proviral excision via a 4-hydroxytamoxifen-inducible Cre recombinase. These findings establish an approach to durable, topically controlled systemic delivery of therapeutic proteins from human skin tissue.
Topics: Administration, Cutaneous; Anemia; Animals; Cell Line; Clobetasol; Dexamethasone; Erythropoietin; Gene Expression Regulation; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; Glucocorticoids; Humans; Integrases; Lentivirus; Mice; Response Elements; Tamoxifen; Viral Proteins
PubMed: 14741782
DOI: 10.1016/j.ymthe.2003.09.016 -
Methods in Enzymology 2006The Rho-associated kinases ROCK I and ROCK II are serine/threonine kinases that play central and critical roles in regulating the actin cytoskeleton. Activation of ROCK...
The Rho-associated kinases ROCK I and ROCK II are serine/threonine kinases that play central and critical roles in regulating the actin cytoskeleton. Activation of ROCK proteins contributes positively to the phosphorylation of myosin II light chains (MLC), myosin ATPase activity, stabilization of filamentous actin, and coupling of the actin-myosin filaments to the plasma membrane, thereby leading to the increased actin-myosin force generation and contractility. We have constructed a conditionally-activated form of ROCK II (called ROCK:ER) by fusing the ROCK II kinase domain to the estrogen receptor hormone-binding domain. In this chapter, we describe the construction and characterization of this regulatable ROCK:ER fusion protein.
Topics: Animals; Cattle; Cell Line; Green Fluorescent Proteins; Humans; Intracellular Signaling Peptides and Proteins; Mice; NIH 3T3 Cells; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Receptors, Estrogen; Recombinant Fusion Proteins; Tamoxifen; rho-Associated Kinases
PubMed: 16472686
DOI: 10.1016/S0076-6879(06)06042-3 -
Science (New York, N.Y.) Aug 2005Mammalian epidermis is maintained by self-renewal of stem cells, but the underlying mechanisms are unknown. Deletion of Rac1, a Rho guanosine triphosphatase, in adult...
Mammalian epidermis is maintained by self-renewal of stem cells, but the underlying mechanisms are unknown. Deletion of Rac1, a Rho guanosine triphosphatase, in adult mouse epidermis stimulated stem cells to divide and undergo terminal differentiation, leading to failure to maintain the interfollicular epidermis, hair follicles, and sebaceous glands. Rac1 exerts its effects in the epidermis by negatively regulating c-Myc through p21-activated kinase 2 (PAK2) phosphorylation. We conclude that a pleiotropic regulator of cell adhesion and the cytoskeleton plays a critical role in controlling exit from the stem cell niche and propose that Rac and Myc represent a global stem cell regulatory axis.
Topics: Animals; Antigens, Differentiation; Apoptosis; Carcinoma, Squamous Cell; Cell Differentiation; Cells, Cultured; Epidermal Cells; Gene Deletion; Humans; Keratinocytes; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neuropeptides; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-myc; Skin Neoplasms; Stem Cells; Tamoxifen; p21-Activated Kinases; rac GTP-Binding Proteins; rac1 GTP-Binding Protein
PubMed: 16081735
DOI: 10.1126/science.1113579 -
FEBS Letters Sep 1996In this study we provide evidence that tamoxifen, the widely used breast cancer drug, is a potent antagonist of glycolipid metabolism. When added to the medium of...
In this study we provide evidence that tamoxifen, the widely used breast cancer drug, is a potent antagonist of glycolipid metabolism. When added to the medium of cultured multidrug resistant (MDR) KB-V-1 carcinoma cells, tamoxifen, at 5.0 microM, drastically lowered the levels of glucosylceramide (glc-cer), as evidenced by a reduction in glc-cer mass. In a similar fashion, in cultured human melanoma cells grown with [3H]galactose, tamoxifen inhibited formation of glc-cer by 44%, and retarded lactosylceramide and ganglioside formation by 50 and 35%, respectively. When glc-cer synthase of melanoma was assayed in cell-free incubations, the inclusion of tamoxifen, at a 1:10 molar ratio with ceramide, inhibited glc-cer synthesis by 50%. These results clearly reveal a new action of tamoxifen and thereby pose intriguing questions regarding mechanisms of action in the realm of estrogen receptor-independent modalities, including effects on MDR.
Topics: Antineoplastic Agents, Hormonal; Chromatography, Thin Layer; Drug Resistance, Multiple; Glucosylceramides; Glucosyltransferases; Glycosphingolipids; Glycosylation; Humans; KB Cells; Melanoma; Molecular Structure; Neoplasms; Tamoxifen; Tumor Cells, Cultured
PubMed: 8843149
DOI: 10.1016/0014-5793(96)00942-8 -
PloS One 2012Krüppel-like factor 1(KLF1) is a hematopoietic-specific zinc finger transcription factor essential for erythroid gene expression. In concert with the transacting factor...
Krüppel-like factor 1(KLF1) is a hematopoietic-specific zinc finger transcription factor essential for erythroid gene expression. In concert with the transacting factor GATA1, KLF1 modulates the coordinate expression of the genes encoding the multi-enzyme heme biosynthetic pathway during erythroid differentiation. To explore the mechanisms underpinning KLF1 action at the gene loci regulating the first 3 steps in this process, we have exploited the K1-ERp erythroid cell line, in which KLF1 translocates rapidly to the nucleus in response to treatment with 4-OH-Tamoxifen (4-OHT). KLF1 acts as a differentiation-independent transcriptional co-regulator of delta-aminolevulinic acid dehydratase (Alad), but not 5-aminolevulinate synthase gene (Alas2) or porphobilinogen deaminase (Pbgd). Similar to its role at the β-globin promoter, KLF1 induces factor recruitment and chromatin changes at the Alad1b promoter in a temporally-specific manner. In contrast to these changes, we observed a distinct mechanism of histone eviction at the Alad1b promoter. Furthermore, KLF1-dependent events were not modulated by GATA1 factor promoter co-occupancy alone. These results not only enhance our understanding of erythroid-specific modulation of heme biosynthetic regulation by KLF1, but provide a model that will facilitate the elucidation of novel KLF1-dependent events at erythroid gene loci that are independent of GATA1 activity.
Topics: Animals; Base Sequence; Cell Line; Chromatin Immunoprecipitation; DNA Primers; Electrophoretic Mobility Shift Assay; Histones; Kruppel-Like Transcription Factors; Mice; Polymerase Chain Reaction; Porphobilinogen Synthase; Promoter Regions, Genetic; RNA Polymerase II; Tamoxifen
PubMed: 23056320
DOI: 10.1371/journal.pone.0046482 -
Nature Cell Biology May 2013Notch signalling is implicated in stem and progenitor cell fate control in numerous organs. Using conditional in vivo genetic labelling we traced the fate of cells...
Notch signalling is implicated in stem and progenitor cell fate control in numerous organs. Using conditional in vivo genetic labelling we traced the fate of cells expressing the Notch2 receptor paralogue and uncovered the existence of two previously unrecognized mammary epithelial cell lineages that we term S (Small) and L (Large). S cells appear in a bead-on-a-string formation and are embedded between the luminal and basal/myoepithelial layers in a unique reiterative pattern, whereas single or paired L cells appear among ductal and alveolar cells. Long-term lineage tracing and functional studies indicate that S and L cells regulate ipsi- and contralateral spatial placement of tertiary branches and formation of alveolar clusters. Our findings revise present models of mammary epithelial cell hierarchy, reveal a hitherto undescribed mechanism regulating branching morphogenesis and may have important implications for identification of the cell-of-origin of distinct breast cancer subtypes.
Topics: Age Factors; Animals; Biomarkers; CD24 Antigen; Cell Differentiation; Cell Lineage; Cell Size; Cytoplasm; Epithelial Cells; Female; Fluorescent Antibody Technique; Lactation; Mammary Glands, Animal; Mice; Mice, Transgenic; Mucin-1; Phenotype; Pregnancy; Receptor, Notch2; Signal Transduction; Staining and Labeling; Tamoxifen; beta-Galactosidase
PubMed: 23604318
DOI: 10.1038/ncb2725 -
Bioorganic & Medicinal Chemistry Oct 2013To generate the first series of prodigiosene conjugates, the tripyrrolic skeleton was appended to estrone, tamoxifen and porphyrin frameworks by way of ester linkers and...
To generate the first series of prodigiosene conjugates, the tripyrrolic skeleton was appended to estrone, tamoxifen and porphyrin frameworks by way of ester linkers and various hydrocarbon chain lengths. The ability of the conjugates to inhibit various types of cancer cells was evaluated in vitro. The porphyrin conjugates did not exhibit significant activity. The estrone conjugates exhibited modest activity, for the most part. However, significantly greater growth inhibition activity against certain breast, colon, lung, leukemia, melanoma and prostate cell lines was noted. This unusual effect for this first generation model class of compound warrants further investigation and comparison to cases where estrogens are linked to prodigiosenes via connection points that do not feature in estrogen receptor binding. The 4-hydroxytamoxifen conjugates exhibit nanomolar range activity against the MCF-7 breast cancer cell line, paving the way to expand the scope and connectivity of prodigiosene-tamoxifen conjugates.
Topics: Cell Line, Tumor; Cell Proliferation; Cell Survival; Estrone; Humans; MCF-7 Cells; Molecular Structure; Neoplasms; Porphyrins; Prodigiosin; Tamoxifen
PubMed: 23958515
DOI: 10.1016/j.bmc.2013.07.042 -
Methods in Molecular Biology (Clifton,... 2011Argonautes (Agos) are core effectors of RNA silencing. In several nonmammalian organisms, multiple Agos are known to exhibit specialized functions for distinct RNA...
Argonautes (Agos) are core effectors of RNA silencing. In several nonmammalian organisms, multiple Agos are known to exhibit specialized functions for distinct RNA silencing pathway. Mammals have four closely related Agos. To examine the functions of mammalian Agos in the microRNA silencing pathway, we generated mouse embryonic stem (ES) cells that are nullizygous for all Agos. This chapter describes a variety of techniques including BAC recombineering, gene targeting, and inducible Cre-loxP recombination, used to generate inducible Ago knock-out ES cells. The Ago-deficient ES cells provide an important tool for the study of mammalian RNA silencing.
Topics: Animals; Cell Line; Chromosomes, Artificial, Bacterial; Embryonic Stem Cells; Estrogen Antagonists; Eukaryotic Initiation Factors; Gene Order; Gene Targeting; Genetic Vectors; Integrases; Mice; Molecular Biology; Promoter Regions, Genetic; Recombination, Genetic; Research Design; Tamoxifen
PubMed: 21528461
DOI: 10.1007/978-1-61779-046-1_19